Cellulose sulfate and other sulfated polysaccharides to prevent and treat papilloma virus infection and other infections

ABSTRACT

A method for treating and preventing various infections, including papilloma virus and fungal and parasitic infections is provided. In particular, an effective amount of a sulfated polysaccharide, such as cellulose sulfate and dextran sulfate are administered to prevent and treat these infections. The invention also relates to use of these compounds for the prevention and inhibition of malignant epithelial lesions associated with papilloma virus, such as cervical cancer.

This application is a provisional of Ser. No. 60/215,325 filed Jun. 30,2000.

FIELD OF THE INVENTION

This invention relates to prevention and treatment of various infectiousagents and in particular, relates to inhibitory activity of cellulosesulfate and other sulfated polysaccharides against various infectiousagents, including papilloma virus and various vaginitis-causingmicrobes.

BACKGROUND

U.S. Pat. No. 4,840,941 (941) describes inhibitory effects of certainsulfated polysaccharides on the enveloped retrovirus, human T-celllymphotrophic virus-III (now known as HIV-1 (human immunodeficiencyvirus-1)). As disclosed in U.S. Pat. No. 5,288,704, sulfatedpolysaccharides are also known to be effective against various otherenveloped viruses and in particular herpes simplex virus (HSV). The 941patent, however, discloses that the inhibitory characteristics ofsulfated polysaccharides against HIV-1 is quite different from theactivities of polysaccharide sulfates against herpes virus. Sincedifferent viruses can have fundamentally different properties, asulfated polysaccharide which is effective against one virus may not beeffective against a different virus.

While the binding of human papilloma virus-like particles (VLP's) toHaCaT cells has been shown to be inhibited by heparin and dextransulfate (Joyce et al. The L1 Major Capsid Protein of HumanPapillomavirus Type 11 Recombinant Virus-like Particles Interacts withHepariin and Cell-surface Glycosaminoglycans oil Human Keratinocytes.The Journal of Biological Chemisty, 1999, Vol 274, No. 9, February 26,pp. 5810–5822), studies with VLP's do not reflect papilloma virusinfection and it is not known that sulfated polysaccharides can inhibitpapilloma virus infection. Papilloma virus differs from HSV and HIV inthat it does not have an envelope and it differs from retroviruses suchas HIV since it is a DNA virus and does not rely on the enzyme reversetranscriptase for replication. This difference may explain theresistance of papilloma virus to nonoxynol-9, a commonly usedspermicide, which has been shown to inhibit both HIV and HSV (Hermonat,P. L., Daniel, R. W. and Shah, K. V. The spermicide nonoxynol-9 does notinactivate papillomavirus Sex. Transm. Dis. 1992; 19:203–205).

Papilloma viruses infect basal cells of epithelia and induce squamousepithelial and fibroepithelial tumors, e.g., warts (papillomas) andcondylomata and can lead to malignant epithelial lesions. (TzenanGiroglou, et al. Human Papillomavirus Infection Requires Cell SurfaceHeparan Sulfate Journal of Virology, February 2001, p. 1565–1570).Genital human papilloma virus infections represent one of the mostfrequent sexually transmitted diseases (STDs) and papilloma virusinfection of the vaginal mucosa in women has been linked to cervicalcancer. Cervical cancer represents the second most frequent cause ofcancer-related deaths in women and accounts for more than 200,000 deathsper year world-wide (Pisani, P., Parkin, D. M., and Ferlay, J. Estimatesof the worldwide mortality from eighteen major cancers in 1985.Implications for prevention and projections of future burden.International Journal of Cancer 55:891–903. 1993).

To date, very few reagents with microbicidal activity against humanpapilloma virus (HPV) infections have been described. These includereagents that specifically target HPVs such as monoclonal antibodieswith virus neutralizing activity (Christensen, N. D., N. M. Cladel, andC. A. Reed. 1995. Postattachment neutralization of papillomaviruses bymonoclonal and polyclonal antibodies. Virology 207:136–142; Christensen,N. D., J. W. Kreider, N. M. Cladel, S. D. Patrick, and P. A. Welsh.1990. Monoclonal antibody-mediated neutralization of infectious humanpapillomavirus type 11. J. Virol. 64:5678–5681) and virus non-specificagents such as povidone-iodine (Sokal, D. C. and P. L. Hermonat. 1995.Inactivation of papillomavirus by low concentrations of povidone-iodine.Sex Transm. Dis. 22:22–24.), alkyl sulfates and monocaprin (Howett, M.K., E. B. Neely, N. D. Christensen, B. Wigdahl, F. C. Krebs, D. Malamud,S. D. Patrick, M. D. Pickel, P. A. Welsh, C. A. Reed, M. G. Ward, L. R.Budgeon, and J. W. Kreider. 1999. A broad-spectrum microbicide withvirucidal activity against sexually transmitted viruses. Antimicrob.Agents Chemother. 43:314–321; Howett, M. K., Wigdahl, B., Malamud, D.,Christensen, N. D., Wyrick, P. B., Krebs, F. C., and Catalone, B. J.Alkyl sulfates: a new family of broad spectrum microbicides. XIIIInternational AIDS Conference, 707–712. 2000. Durban, South Africa,Monduzzi Editore). Several reagents that have microbicidal activityagainst a broad range of STDs have proven to be ineffective againstpapillomaviruses such as C31G and as mentioned above nonoxynol-9. Someof these agents also induce significant cellular cytotoxicity. Aneffective treatment or prevention of papilloma virus infection iscurrently not available.

Cellulose sulfate, a sulfated polysaccharide can be synthesized byvarious known methods of sulfation of cellulose and may be readilyobtained commercially. Sulfated cellulose has been reported to inhibitHIV activities in vitro (Yamamoto et al., Carbohydrate Polymers 14(1990) 53–63). U.S. Pat. No. 6,063,773 (773) discloses the inhibitoryeffects of cellulose sulfate on HIV and HSV and further discloses thatit can be used to treat or prevent bacterial infections. The 773 patentalso discloses cellulose sulfate can reduce the risk of conception.

SUMMARY OF THE INVENTION

The present invention is based in part on the unexpected finding thatcellulose sulfate is effective against papilloma virus infection andagainst other infections including those associated with fungal andparasitic vaginitis. Cellulose sulfate is also effective against manyvaginosis-causing bacteria.

In one aspect, the present invention relates to a method of preventing,inhibiting or treating an infection by papilloma virus in a subject inneed of such prevention, inhibition or treatment comprisingadministering an effective amount of a sulfated polysaccharide such ascellulose sulfate and dextran sulfate. In another aspect, the inventionrelates to a method of preventing or inhibiting a malignant epitheliallesion, including cervical cancer, in a subject in need of suchprevention or inhibition comprising administering an effective amount ofa sulfated polysaccharide such as cellulose sulfate and dextran sulfate.In other aspects, the invention relates to a method of preventing,inhibiting or treating other infections, including fungal and parasiticinfections, such as for example by Trichomonas vaginalis, Aspergillusniger and Candida albicans, in a patient in need of such prevention,inhibition and treatment comprising administering an effective amount ofa sulfated polysaccharide such as cellulose sulfate.

The present invention also relates to use of an effective amount of asulfated polysaccharide such as cellulose sulfate and dextran sulfatefor preventing, inhibiting or treating an infection by papilloma virusin a subject in need of such prevention, inhibition or treatment. Inanother aspect, the invention relates to use of an effective amount of asulfated polysaccharide such as cellulose sulfate and dextran sulfatefor preventing or inhibiting a malignant epithelial lesion, includingcervical cancer, in a subject in need of such prevention or inhibition.In other aspects, the invention relates use of an effective amount of asulfated polysaccharide such as cellulose sulfate for preventing,inhibiting or treating other infections, including fungal and parasiticinfections, such as for example by Trichomonas vaginalis, Aspergillusniger and Candida albicans, in a patient in need of such prevention,inhibition and treatment.

DETAILED DESCRIPTION OF THE INVENTION

It has been found that cellulose sulfate and dextran sulfate areeffective in inhibiting infection by papilloma virus and against fungaland parasitic infections including those associated with vaginitis.Cellulose sulfate is also effective against many vaginosis-causingbacteria. A sulfated polysaccharide such as cellulose sulfate anddextran sulfate therefore can be used to prevent, inhibit or treatinfections caused by these organisms. Moreover, since papilloma virusinfection is associated with malignant epithelial lesions, includingcervical cancer, a sulfated polysaccharide such as cellulose sulfate ordextran sulfate, by preventing papilloma virus infection can alsoprevent these lesions, including cervical cancer. Moreover, since thesecompounds can effectively inactivate papilloma virus, they can inhibitthese lesions, including cervical cancer by inhibiting the spread of theinfectious agent.

Cellulose sulfate of a wide range molecular weight (Mr) may be used. Inone embodiment, cellulose sulfate of average Mr greater than about500,000 daltons may be used. In another embodiment, cellulose sulfate ofaverage Mr of about 1–2 million daltons may be used. The degree ofsulfation of cellulose sulfate is preferably above 12% and mostpreferably about 17–18% which represents maximal sulfation. Cellulosesulfate in the form of a pharmaceutically acceptable salt, for examplesodium cellulose sulfate may also be used. Other pharmaceuticallyacceptable salts include, among others, potassium, lithium and ammoniumcellulose sulfate.

Cellulose sulfate in an effective amount may be administered in asuitable dosage form, depending on the site of administration. An“effective amount” refers to an amount effective at dosages and forperiods of time necessary to achieve the desired therapeutic result,such as to prevent or inhibit infections by papilloma virus or othermicrobes. The effective amount may vary according to various factorssuch as the infection being treated, disease state, age, sex and weightof the individual being treated. While the effective amount can bereadily determined, the studies to date suggest that best results may beachieved with about 0.1 to 200 mg/ml of cellulose sulfate, preferably 1to 100 mg/ml and more preferably 50 to 100 mg/ml.

An effective amount of cellulose sulfate may be administered to the areaor areas that have or are expected to come into contact with theinfectious agent. For example, to prevent, inhibit or treat vaginalinfections, or to prevent or inhibit cervical cancer, cellulose sulfatemay be administered as gels, foams, suppositories, creams or aerosolsinto the vaginal cavity using appropriate applicators. In the case of asexually transmitted infection such as papilloma virus infection,cellulose sulfate may also be administered to the rectum, or usingsuitable edible capsules and flavouring agents, to the mouth, or to thevaginal cavity, of one or more sexual partners, whether known to beinfected or not, to prevent or inhibit transmission during vaginal oranal intercourse or oral sex. Cellulose sulfate may be administeredprior to, during or after sexual activity, providing farther flexibilityand ease of use. If administered after sexual activity, best results maybe achieved immediately following the sexual activity. To prevent,inhibit or treat skin infection, for example, caused by fungalinfection, including by Candida albicans, or to prevent or inhibitmalignant epithelial lesions, cellulose sulfate may be topically appliedto the skin for example as a cream or gel. Cellulose sulfate may also beadministered as an oral dosage form, for example, in the form of atablet.

Suitable carriers or diluents known to those skilled in the art may becombined in the preparation of a suitable dosage form and patientsreceiving the treatment may be monitored for its effectiveness in theknown manner.

In the case of a gel, cellulose sulfate may be combined with glycerinand suitable preservatives such as methylparaben and propylparaben.Other suitable excipients may also be added, for example, a thickeningagent, such as hydroxyethylcellulose. If cellulose sulfate is used onthe skin, it may simply be mixed in water, saline or a bufferingsolution and applied as a gel.

Phase I Safety Study indicates that cellulose sulfate which isnoncytotoxic is better tolerated than nonoxynol-9, a cytotoxic agentfrequently found in spermicidal gels, and as well as or even better thanK-Y Jelly, a lubricant. Cellulose sulfate offers a further advantage inthat irritation by a cytotoxic agent can cause lesions which mayfacilitate infection and the use of cellulose sulfate is not associatedwith such risks of infection.

Dextran sulfate of a wide range of Mr, may be administered as describedfor cellulose sulfate in similar dosage forms. In one embodiment,dextran sulfate of average Mr greater than about 500,000 may be used. Inanother embodiment, the Mr may be about 1–2 million. The effectiveamount may vary according to various factors, including those alreadydescribed and may be readily determined. In one embodiment, about 0.1 to200 mg/ml of dextran may be administered, preferably about 1 to 100mg/ml and more preferably, 50 to 100 mg/ml.

While the use or administration of cellulose sulfate and dextran sulfateaccording to the invention have been described, other sulfatedpolysaccharides may be similarly administered in similar dosage forms inaccordance with the invention. Preferably, the sulfated polysaccharidehas a Mr ranging from about 15000 to 3,000,000. Preferably, the Mr isgreater than about 500,000. The polysaccharide can be a homo- orheteropolysaccharide, preferably a homopolysaccharide, as are cellulosesulfate and dextran sulfate, with monomeric units consisting of eitheraldo-, deoxyaldo-, keto- or deoxyketopentoses, including, but notrestricted to, arabinose, ribose, deoxyribose, galactose, fructose,sorbose, rhamnose and fucose, joined by either alpha- or beta-linkages.The polymer can be linear or branched, with free hydroxyl groups of themonomeric units maximally or partially sulfated. Preferably, thehydroxyl groups are maximally sulfated. The monomeric units may befurther modified by the presence of carboxyl, amino and ester groups.Examples of suitable sulfated polysaccharides include dermatan sulfate,chondroitin sulfate, pentosan sulfate, fucoidin, mannan sulfate,carrageenan, dextrin sulfate, curdlan sulfate, chitin sulfate, heparinand heparin sulfate all of which may be obtained commercially.

The terms, cellulose sulfate, dextran sulfate, dermatan sulfate,chondroitin sulfate, pentosan sulfate, fucoidin, mannan sulfate,carrageenan, dextrin sulfate, curdlan sulfate, and chitin sulfate areintended include within their scope pharmaceutically acceptable saltsthereof. Similarly, the term sulfated polysaccharides include within itsscope pharmaceutically acceptable salts thereof. Moreover, while humanpatients are contemplated as subjects in need of prevention, inhibitionor treatment according to the invention, other mammals susceptible tosimilar infection (and lesions in the case of infection by papillomavirus) are also subjects for such prevention, inhibition or treatment.

EXAMPLE 1

Inhibition of Bovine Papilloma Virus (BPV)

Cellulose sulfate was tested for its ability to inhibit BPV infection bycell focus formation assay (see Hermonat et al. (1992) for a descriptionof this assay). The results are shown below. Cellulose sulfate fromDextran Products Limited (Lot 80971 in the form of sodium cellulosesulfate, known as Ushercell J.) was mixed with BPV type I (obtained frombovine fibropapillomas) prior to adding the virus to mouse fibroblastline C127 cells or mixed with these host cells first prior to adding thevirus. In one molecular weight study, the Mr range of cellulose sulfatewas about 750 to 20.3 million, with an average Mr of about 1.01 million.The peak Mr as seen on HPLC was about 2.77 million. In another study,the average molecular weight was determined to be about 1.9 million witha peak Mr of about 2.3 million. Unless otherwise specified, the Mr is indaltons.

TABLE A Inhibition of Bovine Papillomavirus type I by Cellulose sulfateMethod of Compound Exposure [Cellulose Pre-incubated with virus priorPre-incubated with host cells sulfate] to addition to host cells priorto addition to virus (μg/ml) Viral-induced foci per cultureViral-induced foci per culture 0 450 450 450 450 5.0 121  72  97  60 50 37  10  32  12 500  0^(A)  0^(A)  0  0 5000  0^(B)  0^(B)  0  0^(A)Mild monolayer disruption ^(B)Monolayer at approximately 80%confluency; disrupted

The results indicate a dose response and the formation of oncogenic fociby the virus is completely inhibited at 500 μg/ml when cellulose sulfateis mixed with the virus or with the host cells.

The assay was repeated using different concentrations of cellulosesulfate and the results are shown below.

TABLE B Inhibition of Bovine Papillomavirus type I by Cellulose sulfateMethod of Compound Exposure [Cellulose Pre-incubated with virus priorPre-incubated with host cells sulfate] to addition to host cells priorto addition to virus (μg/ml) Viral-induced foci per cultureViral-induced foci per culture 0 240 240 196 196 1.6 196  53 168  13 8.0 60  5 124  3 40 116  0 104  0 200  34  0  42  0

Complete inhibition was seen at 40 μg/ml only when cellulose sulfate waspre-incubated with host cells, although partial inhibition was noted atthat dose level when mixed first with the virus. At 200 μg/ml, almostcomplete inhibition of infection was obtained when cellulose sulfate waspre-incubated with the virus before addition to the host cells. Theseresults show that cellulose sulfate inhibits infection both when addedfirst to the virus or first to the host cells although it tends to besomewhat more effective when added first to the host cells.

In a similar study using the BPV-1 focus forming assay, the effect ofthe cellulose sulfate and dextran sulfate on BPV was tested. In thisstudy, cellulose sulfate tested was as described above. Dextran sulfateused was from Dextran Products Limited (Lot DSM-122) prepared usingdextran of average Mr of about 500,000 (based on viscosity) and isestimated to have a final average Mr greater than about 500,000 and maybe about 1 to 1.1 million.

Microbicidal activity of the compounds was tested using thewell-characterized BPV-1 focus-forming assay (Dvoretzky, I., R. Shober,S. K. Chattopadhyay, and D. R. Lowy. 1980. A quantitative in vitro focusassay for bovine papilloma virus. Virology 103:369–375), withmodifications for microbicide testing (Hermonat, P. L., (1992) supra;Howett, M. K., et al. (1999) supra. The terms inhibiting or microbicidalactivity when used are intended to refer broadly to microbe infectionand/or microbe inactivating effect.

Aliquots of BPV-1 containing approximately 100–200 focus-forming unitswere preincubated with dilutions of compounds for 10 min at 37° C. priorto addition to cultures of mouse C127 cells. Cultures of C127 cells wereset up in T25 tissue culture flasks (Corning, N.Y.), containing 3×10⁵cells per flask. Virus-compound mixtures in a total of 50 μl were thenadded to flasks in 1 ml of media, and an additional 3 ml of media addedafter 24 hrs culture. Media was changed every 3–4 days for a period of 2weeks. Foci were enumerated following staining of the monolayer withcrystal violet and counting stained foci microscopically. Eachconcentration of compound was tested in duplicate, and the mean ±SD offoci number for the preincubation virus-drug concentration for eachcompound is shown below as Table C.

TABLE C Inhibitory effect of cellulose sulfate and dextran sulfate whenmixed with the bovine papilloma virus before addition of the mixture tohost cells Average ± standard deviation (% of control) Cellulose sulfateDextran sulfate Concentration C127 cell C127-D10 C127 cell C127-D10(μg/ml) line clone line clone 0 (control) 100 100 100 100 0.01  80 ± 12 73 ± 31  81 ± 3  64 ± 8.0 0.1  76 ± 9  80 ± 16 102 ± 10  83 ± 16 1  73± 5  33 ± 3  97 ± 7  69 ± 7 10  42 ± 3  3 ± 3 113 ± 2  3 ± 3 100  10 ± 2 2 ± 3  43 ± 7  3 ± 3 1,000  0  0  6 ± 3  0 10,000  0  0  0  0

Microbicidal activity of compounds was also tested by pre-incubation ofcells with compounds followed by addition of virus to compound-coatedC127 cells. In these experiments, dilutions of compounds were added tocultures of C127 cells, incubated for 1 hr at 37° C., washed 3 timeswith media to remove unbound compound, prior to addition ofapproximately 100 focus-forming units of BPV-1. The cultures wereincubated for an additional hour, washed three times to remove unboundvirus, then the incubation was continued for two weeks with mediachanges every 3–4 days and foci counted as described above. The resultsare shown below in Table D.

TABLE D Inhibitory effect of cellulose sulfate and dextran sulfate whenmixed with target cells, followed by washing of the cells and additionof the bovine papilloma virus Average ± standard deviation (foci/ well)Concentration (μg/ml) Cellulose sulfate Dextran sulfate 0  88 ± 15.6 115(n = 1) 10 103 ± 6.3  31 ± 16.9 100 120 ± 8.4  15 ± 4.6 1,000  87 ± 45.1 4 ± 2.6 10,000  2 ± 1.3  0

The results from Table C demonstrated that both compounds showedmicrobicidal activity against BPV-1. From 10 to 100 μg/ml CS showedmoderate to high inhibition of papilloma virus infectivity using theC127 cell line, with complete inhibition at 1 mg/ml. DS showed moderateinhibition at 100 μg/ml and very high inhibition at 1 mg/ml. Clones werederived from the parental C127 cell line because of the consistentfailure of BPV-1 to induce foci following several cell passages of theuncloned parental cell line. One clone, labeled C127-D10, which producedfoci upon BPV-1 infection, was chosen for a repeat testing of thecompounds. When this clone was tested for microbicidal activity, lesscompound was required to achieve high reduction in BPV-1-induced fociwhen compared to the uncloned parental-C 127 cells.

Pre-incubation of C127-D10 cells with compounds prior to addition ofBPV-1 was tested to determine whether the microbicidal effects of CS andDS extended to a blockage of virus interaction with cell surfaces. Inthese experiments, titrations of compounds were added to cell culturesfollowed by washing away unbound reagent prior to addition of virus.After a one-hour incubation with virus, unbound virus was removed bywashing and the cultures monitored for foci after two weeks.

The results (Table D) indicated that these reagents showed someinterference of virus with host cell surfaces as evidenced by adose-dependent reduction of BPV-1-induced foci. DS showed strongerinterference, with substantial reduction in foci at doses of 10 μg/ml.In contrast, CS showed only weak microbicidal effects when C127-D10cells were pre-treated with this compound except at a concentration of10 mg/ml. The difference in the results seen with the earlier study islikely due to the fact that in the earlier study, unbound cellulosesulfate was not washed away prior to addition of virus. Since cellulosesulfate or other sulfated polysaccharide upon administration, forexample, vaginally should remain in the vaginal cavity, the earlierresults more likely represents in vivo effects and the compound in vivois expected to inactivate papilloma virus both by direct association andby interfering with virus attachment to cells.

EXAMPLE 2

Inhibition of Human Papilloma Virus (HPV)

Cellulose sulfate and dextran sulfate were each prepared as a 2 mg/mlsolution in 0.9% NaCl and tested for microbicidal activity using the invitro HPV transient infection assay originally described by Smith andcolleagues (Smith, L. H., C. Foster, M. E. Hitchcock, and R. Isseroff.1993. In vitro HPFV-11 infection of human foreskin. J. Invest. Dermatol.101:292–295) with some modifications (Ludmerer, S. W., W. L. McClements,X. M. Wang, J. C. Ling, K. U. Jansen, and N. D. Christensen. 2000. HPV11mutant virus-like particles elicit immune responses that neutralizevirus and delineate a novel neutralizing domain. Virology 266:237–245).An ELISA-based read-out of Optical Density (OD) values using alkalinephosphatase cleavage of the substrate p-nitrophenyl phosphate was alsoused to measure HPV infection as described below.

In the standard RT-PCR assay (Ludmerer, S. W., et al. supra; Smith, L.H., et al. supra; Smith, L. H., C. Foster, M. E. Hitchcock, G. S.Leiserowitz, K. Hall, R. Isseroff, N. D. Christensen, and J. W. Kreider.1995. Titration of HPV-11 infectivity and antibody neutralization can bemeasured in vitro. J. Invest. Dermatol. 105:438–444) for detection ofHPV-11 infection, aliquots of HPV-11 (10 μl) were preincubated withdilutions of compounds (40 μl) for 30 min at 37° C. then the mixtureswere added to cultures of human A431 cells. Replicate cultures of A431cells were set up by plating 5×10⁵ cells (in 1 ml tissue culture medium)per well into 6-well culture plates. Virus-compound mixtures were addedto individual A431 cultures and the cultures were incubated for afurther 4 days (after overnight incubation, an additional 2 ml ofculture medium was added to each culture). Cells were harvested in 1 mlTrizol (GIBCO/BRL), then total RNA prepared for RT and production ofviral cDNA from spliced viral transcripts spanning a major splice sitebetween E1 and E4 (Ludmerer, S. W. et al. supra; Smith, L. H. et al.supra). Two rounds of PCR amplification using nested primers preparedfrom the published sequence were conducted for detection of the splicedviral transcript, and the PCR products were detected as ethidium-stainedbands on agarose gels (Ludmerer, S. W. et al. supra; Smith, L. H. et al.supra). PCR products were cloned and sequenced to confirm the viralorigin of the PCR product. The presence of the correct sized viral PCRproduct was used to confirm successful infection by HPV-11, as well as afailure to inactivate and/or block the virus by the test compound. Incontrast, the lack of a viral PCR product was interpreted to indicatevirus inactivation, and/or a failure of the virus to infect A431 cells.Amplified B-actin transcripts (Smith, L. H., et al. supra) were used asa control to establish the integrity of RNA isolation and RT-PCRprocedures for uninfected cells and for cultures in which HPV-11inactivation was achieved.

The RT-PCR assay to detect HPV-40 infection was designed similarly forthe detection of HPV-11 infection as described above.

A modification of the RT-PCR assay that incorporates an ELISA-basedread-out (Boehringer-Mannheim) was also included to assess microbicidalactivity. Replicate cell cultures of A431 cells were infected with analiquot of infectious HPV virions as described above. After 4 days ofculture, cells were harvested and RNA extracted. RNA was subjected to RTusing downstream anti-sense (reverse) primers for HPV-11 or HPV-40 andβ-actin (as a control/housekeeping cellular transcript) to initiate cDNAsynthesis. The cDNA was processed through 2 sets of 30 cycles of PCRamplification using nested primers: the second set of cycles useddigoxygenin (DIG)-dUTP to label the PCR products with DIG. DIG-labeledPCR products were denatured then renatured together with a biotinylatedoligonucleotide specific for the targeted PCR product. Biotinylatedproducts were detected in ELISA with plates coated with streptavidin (tocapture the biotinylated target PCR product) then anti-DIG antibody andsubstrate. Labeled PCR products were added directly to ELISA plates, ortitrated at 10-fold dilutions in duplicate for each cell culture foreach virus dilution.

TABLE E ELISA RT-PCR detection of transient infection of HPV-11 andHPV-40. Mean Cell culture Concentration of PCR (SD) of ELISA O.D.readings conditions^(a) products^(b) HPV-11 probe HPV-40 probe HPV-11 101.827 (0.174) −0.013 (0.000) infection 1 1.540 (0.034) NT^(c) 0.1 0.845(0.039) NT HPV-40 10 0.012 (0.002)  2.000 (0.000) infection 1 NT  1.842(0.080) 0.1 NT  1.027 (0.028) ^(a)A431 cultures infected with eitherHPV-11 or HPV-40. ^(b)Volume (μl) of reaction products from the secondset of PCR amplification products added to the ELISA wells. ^(c)Nottested.

TABLE F RT-PCR ELISA for detection of transient infection of human A431cells with HPV-11 or HPV-40.^(a) Compound (μg/ml at viral Mean (SD) ofO.D. reading for RT-PCR ELISA pretreatment dose) HPV-11 probe β-actinprobe Experiment #1^(b) Cells alone 0.043 (0.004) 1.645 (0.052) HPV-11only 1.417 (0.063) 1.564 (0.051) CS 1000 μg/ml (no 0.028 (0.004) 1.427(0.013) virus) DS 1000 μg/ml (no 0.000 (0.000) 1.470 (0.001) virus)^(c)Cs 1000 μg/ml 0.038 (0.001) 1.518 (0.020) ^(c)CS 100 μg/ml 0.049(0.001) 1.532 (0.025) ^(c)CS 10 μg/ml 1.485 (0.045) 1.576 (0.021) ^(c)DS100 μg/ml 0.035 (0.000) 1.583 (0.022) ^(c)DS 10 μg/ml 0.023 (0.000)1.636 (0.105) HPV-40 probe β-actin probe Experiment #2^(d) ^(c)CS 1000μg/ml 0.037 (0.004) N.D.^(e) ^(c)CS 100 μg/ml 0.128 (0.008) N.D. ^(c)CS10 μg/ml 0.397 (0.050) N.D. ^(c)DS 1000 μg/ml 0.006 (0.007) N.D. ^(c)DS100 μg/ml 0.375 (0.073) N.D. ^(c)DS 10 μg/ml 0.042 (0.007) N.D. HPV-40only 1.320 (0.074) N.D. ^(a)Two additional experiments yielded similarresults. ^(b)Infected with HPV-11. ^(c)With virus. ^(d)Infected withHPV-40. ^(e)Not determined.

The microbicidal activity was assessed either as the detection ofethidium stained PCR products or as an ELISA-based read-out as describedabove. Virus inactivation or lack of virus infection was evidenced bythe failure to detect viral spliced RT-PCR products (results not shown)and/or the lack of ELISA values above background when using the ELISAassay to detect labeled PCR products. An initial experiment wasconducted to test the specificity of the ELISA-based RT-PCR assay usingHPV-11 and HPV-40 infection of A431 cells (Table E). Highly specificdetection of either HPV-11 or -40 was observed by the presence of highELISA O.D. values for the HPV-11 probe from HPV-11-infected but notHPV-40-infected cultures and vice versa.

Both compounds demonstrated strong microbicidal activity against bothHPV-11 and -40 in both tests and representative experiments using RT-PCTElisa are summarized in Table F. The results showed that RT-PCR productsfrom cells alone or from uninfected cultures treated with compoundsconsistently demonstrated low O.D. readings in the ELISA assay for theHPV products and high O.D. readings for the β-actin product. Upon HPV-11and/or HPV-40 infection, cultures showed high levels of ELISA detectableviral products, and addition of microbicides decreased the signal backto background (uninfected) levels. For CS, this occurred at 100 and 1000μg/ml, and for DS at 10 and 100 μg/ml when tested for microbicidalactivity against HPV-11. In assays for HPV-40 infectivity, CS wasmicrobicidal at 100 and 1000 μg/ml and DS at 10 and 1000 μg/ml. Therewas no cellular cytotoxicity for any of the doses of compounds asdetermined by microscopic examination of the cell cultures.

Cellulose sulfate (CS) described in Example 1 was tested in each of thefollowing examples.

EXAMPLE 3

Inhibition of Trichomonas by Cellulose Sulfate

The inhibitory effect of CS on trichomonas vaginalis, protozoa known tocause vaginitis, is shown below. The organisms were grown in modifiedDiamond's medium. CS was mixed with the organism in modified Diamond'smedium at a final concentration of about 5 mg/ml (5.12 mg/ml) andincubated anaerobically at 35° C. Samples were collected at various timepoints and the number of live trichomonas counted with a hemacytomer.The same procedure was performed in the absence of CS. The volume of theinoculum was varied as indicated in the Tables to study the effect ofincreasing amounts of the organism on the results.

TABLE G Control Incubation time 400 μl Trich 200 μl Trich 100 μl Trich50 μl Trich 16 hr 260 130  55  25 24 hr 620 210 100  60 40 hr Tntc 850360 130 48 hr Tntc tntc 500 250 Numbers indicate the number of lifeorganisms per ml Trich = Trichomonas Tntc = too numerous to count

TABLE H 5 mg/ml Cellulose Sulfate Incubation time 400 μl Trich 200 μlTrich 100 μl Trich 50 μl Trich 16 hr 0 0 0 0 24 hr 0 0 0 0 40 hr 0 0 0 048 hr 0 0 0 0

Complete inhibition of growth of the trichomonas culture was observedwhen mixed with 5 mg/ml CS.

A sulfated polysaccharide such as cellulose sulfate, therefore may beused to prevent, inhibit or treat parasitic infection such as byTrichomonas vaginalis and by Enterobius vermicularis also known to causevaginitis and related parasites.

EXAMPLE 4

Inhibition of Fungal, Yeast and Bacterial Growth

6% CS was prepared in water as a translucent gel. Twenty grams of thegel was placed in a plastic screw-cap centrifuge tube. The microbialinoculants were prepared, gently vortexed and 0.1 ml inoculant wasaseptically pipetted into the 20.0 gram gel. This procedure was repeatedfor each microbial organism. The samples were incubated at roomtemperature (20–25° C.) for 14 days or 28 days. The microbes tested andtheir theoretical yields can be found in Table F. After 14 or 28 daysincubation, dilutions (10⁻¹, 10⁻², 10⁻³) of the gels were made in salineand 1 ml plated onto Sabouraud dextrose (Aspergillus and Candida) oronto tryptic soy (other microbes). The number of organisms that grewafter 3–7 days at room temperature were counted.

TABLE I Theoretical* Day Test Organism ATCC# Yield CFU/g Day 14 CFU/g 28CFU/g Aspergillus niger 16404 2.3 × 10⁵ 0 0 Candida albicans 10231 4.5 ×10⁵ 0 0 Staphylococcus 6538 1.2 × 10⁵ 0 0 aureus Eschericia coli 87392.0 × 10⁵ 0 0 Pseudomonas 9027 3.0 × 10⁵ 0 0 aeruginosa *Theoreticalyield = CFU/ml × amount of inoculant (.1 ml)/amount of sodium cellulosesulfate gel (20 g)

The results shows surprisingly that CS inhibits fungal and yeastinfections, including Candida which is associated with vaginitis. CSpreviously shown to inhibit N. goizorrlzea and C. trachoinatisinfections also showed inhibition of bacteria S. aureus., E. coli and P.aerugiizosa.

In another study, CS was dissolved in water at the concentrationsindicated below. No other ingredients, including preservatives, wereadded. The gels were challenged with microbes as described above exceptthat the gels were examined for the presence of microbes after 1, 2, 3,4 and 7 days. The results were as follows. The amount of microbesorigially inoculated in the gels is also shown below.

Asperigillus niger (CFU × 10⁵/ml) CS % 24 hours 48 hours 72 hours 96hours 7 days 0.06 0 0 0 0 0 0.6 0 0 0 0 0 1.2 0 0 0 0 0 6 0 0 0 0 0 CSgel in as low a concentration as 0.6% completely inactivatedAsperigillus.

Candida albicans (CFU × 10⁵/ml) CS % 24 hours 48 hours 72 hours 96 hours7 days 0.06 0 0 0 0 0 0.6 0 0 0.62 0.655 0.535 1.2 0 0 0 0 0 6 0 0 0 0 0CS gel in as low a concentration as 0.6% inactivated Candida. Completeinactivation was obtained at 1.2 and 6%.

Staphylococcus aureus (CFU × 10⁵/ml) CS % 24 hours 48 hours 72 hours 96hours 7 days 0.06 0.625 0.715 1.04 2.06 5.815 0.6 5.17 5.53 4.82 6.413.25 1.2 2.71 4.725 2.39 3.96 3.55 6 0 0.005 0 0 0 6% CS gel completelyinactivated Staphyloccus. At lower CS concentrations, the microbe wasnot inactivated but growth was prevented. The 0.06% CS gel initiallyinactivated most Staphylococcus (in contrast to the 0.6% and 1.2% gels)but allowed slow growth thereafter.

Escherichia coli (CFU × 10⁵/ml) CS % 24 hours 48 hours 72 hours 96 hours7 days 0.06 0 1.15 1.21 0.13 1.12 0.6 5.6 5.81 4.43 6.92 4.39 1.2 0.983.125 1.625 4.54 3.78 6 0 0.01 0 0 0 6% CS gel completely inactivatedEscherichia. At lower CS concentrations, the microbe was either notinactivated or only to a minor extent (except by the 0.06% CS gel) butno growth occurs.

Pseudomans aeruginosa (CFU × 10⁵/ml) CS % 24 hours 48 hours 72 hours 96hours 7 days 0.06 0 0.48 1.125 1.87 2.04 0.6 5.43 6.80 4.43 6.92 6.051.2 2.08 2.625 2.31 2.82 4.79 6 0 0 0 0 0 6% CS gel completelyinactivated Pseudomonas. At lower CS concentrations, the microbe was notinactivated but growth was prevented. The 0.06% CS gel initiallyinactivated most Staphylococcus (in contrast to the 0.6% and 1.2% gels)but allowed slow growth thereafter.

Original inoculum (CFU × 10⁵/ml) Asperigillus niger 2.25 Candidaalbicans 4.55 Staphylococcus aureus 3.78 Escherichia coli 5.98Pseudomonas aeruginosa 2.23

A sulfated polysaccharide, such as cellulose sulfate therefore may beused to prevent, inhibit or treat fungal infections, such as by Candidaand Asperigillus, Trichophyton, Epidernophyton and Microsporum,including C. albicans, A. niger, T. pedis, T. cruris and T. capitis andrelated fungal infections.

EXAMPLE 6

Experimental Procedures

A. Broth Method

CS was serially diluted in brucella broth supplemented with laked sheepblood, vitamin K, and hemin (highest concentration tested was 10 mg/ml).Aliquots (1 ml) of the mixtures were then added to 12×75 mm plastictubes. Test strains were suspended in each of the tubes to a turbidityequal to the ½ McFarland Standard in supplemented brucella broth anddiluted about 1:50 in brucella broth to obtain a concentration of about3×10⁶ CFU/ml. Samples (1 ml) of the test strains were added to the CSdilution series and then incubated for 2 days at 37° C. under anaerobicconditions. Tubes containing no organism or no drug were testedsimultaneously as negative and positive controls. For each organismtested, the lowest concentration of CS that completely inhibited growth(MIC) was determined.

B. Agar Method

Various concentrations of cellulose sulfate in water (highestconcentration tested was 0.625 mg/ml) were mixed with molten brucellaagar supplemented with sheep blood, vitamin K, and hemin (NCCLSreference agar dilution method) and then poured into separate plates.After the plates were solidified and dried, suspensions of the testorganisms (10⁸ CFU/ml) were spotted on the surface using a replicatingdevice that delivered a final concentration of 10⁵ CFU/spot. Afterincubation at 37° C. for 48 hours under anaerobic conditions, the plateswere examined for growth. For each organism tested, the lowestconcentration of CS that completely inhibited growth (IC) as compared tothe drug free growth control plate was determined.

Results

The results are shown in Table J. At the time the agar method was used,only concentrations up to 0.625 mg/ml of cellulose sulfate were testedso that the agar method results are limited. The broth method mayrepresent a better indicator of activity because the movement of largemolecules is more restricted in agar.

In the broth method, CS inhibited both strains of Fusobacteriumnucleatum and both strains of Fusobacterium gonidiaformans.

Neither one of the strains of Prevotella melaminogenica were inhibitedat the concentrations tested. However, all strains of Prevotellaintermedia, Prevotella bivia and Prevotella disiens were inhibited byCS.

Two strains of Porphyromonas asaccharolytica were inhibited but a thirdone was not at the concentrations tested. Both strains of Porphyromonaslevii were inhibited.

CS inhibited both strains of Gardnerella vaginalis.

All strains of Peptostreptococcus magnus, Peptostreptococcus tetradiusand Peptostreptococcus asaccharolyticus were inhibited by CS.

Both strains of Eubacterium lentum were inhibited.

CS inhibited one strain of Clostridiun perfringes but not another one atthe concentrations tested.

One strain of Bacteroides thetaiotaomicron was inhibited but threeothers were not at the concentrations tested. Similarly, one strain ofBacteroides fragilis was inhibited but three others were not at theconcentrations tested.

TABLE J INHIBITION OF BV-CAUSING MICROBES BY CELLULOSE SULFATE (LOT80971) Broth Broth (study 1) (study 2) CS CS Agar* MIC MIC CS rma#Organism (mg/ml) (mg/ml) MIC (mg/ml) 10481 F. nucleatum 5 >0.625** 11518F. nucleatum 10 >0.625 11423 F. gonidiaformans 5 >0.625 11653 F.gonidiaformans 5 0.156 9052 Prev. melaninogenica NI >0.625 5657 Prev.melaninogenica NI >0.625 11142 Prev. intermedia 10 >0.625 11168 Prev.intermedia 10 >0.625 11697 Prev. bivia 0.6 5 >0.625 11683 Prev. bivia NI5 >0.625 11579 Prev. disiens 10 >0.625 11698 Prev. disiens 10 >0.62511690 Porph. asacch. NI 11656 Porph. asacch. 5 >0.625 11612 Porph.asacch. 0.08 11425 Porph. levii 0.6 0.6 >0.625 11601 Porph. levii 0.30.3 0.04 12066 Gard. vaginalis 5 12262 Gard. vaginalis 5 *Onlyconcentrations of 0.6 mg/ml or less were tested in agar **i.e. No effectconcentration up to 0.625 mg/ml NI for the broth method = not inhibitedby 10 mg/ml (the highest concentration tested) Blank = not tested

TABLE J INHIBITION OF BV-CAUSING MICROBES BY CELLULOSE SULFATE (LOT80971) (cont.) Agar* Broth (study 1) Broth (study 2) CS CS CS MIC rma#Organism MIC (mg/ml) MIC (mg/ml) (mg/ml) 11658 Ps. Magnus 5 >0.625**11598 Ps. Magnus 5 >0.625 11287 Ps. Tetradius 5 >0.625 11253 Ps.Tetradius 5 >0.625 11587 Ps. Asacch. 10 >0.625 11607 Ps. Asacch.10 >0.625 9420 Eubact. Lentum 5 >0.625 11700 Eubact. Lentum 10 >0.62511608 Clost. Perfringes 10 >0.625 11655 Clost. Perfringes NI >0.625 ATCCB. theta NI NI >0.625 ATCC B. theta 10 11604 B. theta NI >0.625 11651 B.theta NI >0.625 ATCC B. fragilis NI 10 >0.625 ATCC B. fragilis NI 11647B. fragilis NI >0.625 11652 B. fragilis NI >0.625 *Only concentrationsof 0.6 mg/ml or less were tested in agar **i.e. No effect concentrationup to 0.625 mg/ml NI = not inhibited by 10 mg/ml (the highestconcentration tested) Blank = not tested

Microbes Tested (see Tables)

-   F. nucleatum=Fusobacterium nucleatum-   F. gonidiaformans=Fusobacterium gonidiaformans-   Prev. melaminogenica=Prevotella melaminogenica-   Prev. intermedia=Prevotella intermedia-   Prev. bivia=Prevotella bivia-   Prev. disiens=Prevotella disiens-   Porph. asacch.=Porphyromonas asaccharolytica-   Porph levii=Porphyromonas levii-   Gard. vaginalis=Gardnerella vaginalis-   Ps. magnus=Peptostreptococcus magnus-   Ps. tetradius=Peptostreptococcus tetradius-   Ps. asacch.=Peptostreptococcus asaccharolyticus-   Eubact. lentum=Eubacterium lentuni-   Clost. perfringes=and Clostridium perfringes-   B. theta=Bacteroides thetaiotaomicron-   B. fragilis=Bacteroides fragilis

EXAMPLE 7

Inhibition of Gardnerella vaginalis

A fresh subculture of G. Vaginalis which is bacteria frequentlyassociated with vaginitis was obtained after overnight growth (16 hours)on a V-agar plate and suspended in sterile phosphate buffered saline(PBS; pH 7.2) to achieve a turbidity of 0.5 (McFarland standard;approximately 108 CFU/ml). The suspension was diluted 10 fold andapplied to HBT bilayer agar plates by swabbing the entire plate. Theplate was allowed to dry for 2–3 min and small wells punched in theagar, 10 mm in diameter. Samples of cellulose sulfate were dissolved at10 mg/ml in PBS and 0.2 ml placed in the agar wells. As control, 0.2 mlPBS was placed in one of the wells. Growth inhibition was indicated by alight area around the well. No growth inhibition was observed with PBS,whereas an area of 6 mm in diameter was found around the cellulosesulfate well, showing growth inhibition.

The studies were repeated using various concentrations of cellulosesulfate (ranging from 10 mg/ml to 0.125 mg/ml) and 4 differentGardnerella vaginalis strains. Dose dependent inhibition of each strainwas observed. The lowest initial concentration of cellulose sulfate atwhich growth inhibition occurred was 0.5 mg/ml.

EXAMPLE 8

Inhibition of Vaginosis-Causing Bacteria

In another study, cellulose sulfate was prepared at a variety ofconcentrations in water (highest tested was 625 μg/ml) and mixed withmolten brucella agar supplemented with sheep blood, vitamin K and hemin.After the plates were poured and dried, suspensions of the test strainswere prepared and applied to the surface of the plates at a finalconcentration of 100,000 CFU per spot. After incubation for 48 hours inan anaerobic environment, the plates were examined for growth and thelowest concentration of compound that inhibited growth determined.

Inhibition of the Following Organisms was Observed:

-   1. Fusobacterium gonidadormans—inhibited at 156 μg/ml and higher-   2. Porphyromonas asacch—inhibited at 80 μg/ml and higher-   3. Porphyromonas levii—inhibited at 40 μg/ml and higher

All references cited herein are fully incorporated by reference. Havingnow described the invention, it will be understood by those skilled inthe art that various modifications can be made to the describedembodiments without departing from the scope and spirit of theinvention. Such modifications are intended to be within the scope of theinvention.

1. A method of treating papilloma virus infection in a subject in needof such treatment comprising administering an effective amount of asulfated polysaccharide to the subject wherein the sulfatedpolysaccharide is cellulose sulfate, dextran sulfate, dermatan sulfate,chondroitin sulfate, pentosan sulfate, fucoidin, mannan sulfate, dextrinsulfate, curdlan sulfate, chitin sulfate, heparin or heparin sulfate. 2.The method according to claim 1 wherein the subject is a human patient.3. The method according to claim 2 wherein the sulfated polysaccharideis cellulose sulfate.
 4. The method according to claim 3 whereinsulfation of cellulose sulfate is at least 12%.
 5. The method accordingto claim 4 wherein cellulose sulfate is maximally sulfated.
 6. Themethod of according to claim 3 wherein cellulose sulfate has an averagemolecular weight (Mr) greater than about 500,000 daltons.
 7. The methodaccording to claim 6 wherein cellulose sulfate has an average Mr ofabout 1–2 million daltons.
 8. The method according to claim 3 whereinthe effective amount is about 0.1 to 200 mg/ml.
 9. The method accordingto claim 8 wherein the effective amount is about 1 to 100 mg/ml.
 10. Themethod according to claim 9 wherein the effective amount is about 50 to100 mg/ml.
 11. The method according to claim 3 wherein cellulose sulfateis administered in combination with a pharmaceutically acceptablecarrier or diluent.
 12. The method according to claim 11 whereincellulose sulfate is administered vaginally.
 13. The method according toclaim 12 wherein said papilloma virus infection results in a malignantepithelial lesion.
 14. The method according to claim 13 wherein saidmalignant epithelial lesion is cervical cancer.
 15. The method accordingto claim 2 wherein the sulfated polysaccharide is dextran sulfate. 16.The method according to claim 15 wherein dextran sulfate has an averageMr greater than about 500,000 daltons.
 17. The method according to claim15 wherein the effective amount is about 0.1 to 200 mg/ml.
 18. Themethod according to claim 17 wherein the effective amount is about 1 to100 mg/ml.
 19. The method according to claim 18 wherein the effectiveamount is about 50 to 100 mg/ml.
 20. The method according to claim 15wherein dextran sulfate is administered in combination with apharmaceutically acceptable carrier or diluent.
 21. The method accordingto claim 20 wherein dextran sulfate is administered vaginally.
 22. Amethod of inhibiting papilloma virus infection in a subject in need ofsuch inhibition comprising administering an effective amount of asulfated polysaccharide to the subject wherein the sulfatedpolysaccharide is cellulose sulfate, dextran sulfate, dermatan sulfate,chondroitin sulfate, pentosan sulfate, fucoidin, mannan sulfate, dextrinsulfate, curdlan sulfate, chitin sulfate, heparin or heparin sulfate.23. The method according to claim 22 wherein the subject is a humanpatient.
 24. The method according to claim 23 wherein the sulfatedpolysaccharide is cellulose sulfate.
 25. The method according to claim24 wherein sulfation of cellulose sulfate is at least 12%.
 26. Themethod according to claim 25 wherein cellulose sulfate is maximallysulfated.
 27. The method according to claim 24 wherein cellulose sulfatehas an average molecular weight (Mr) greater than about 500,000 daltons.28. The method according to claim 27 wherein cellulose sulfate has anaverage Mr of about 1–2 million daltons.
 29. The method according toclaim 24 wherein the effective amount is about 0.1 to 200 mg/ml.
 30. Themethod according to claim 29 wherein the effective amount is about 1 to100 mg/ml.
 31. The method according to claim 30 wherein the effectiveamount is about 50 to 100 mg/ml.
 32. The method according to claim 24wherein cellulose sulfate is administered in combination with apharmaceutically acceptable carrier or diluent.
 33. The method accordingto claim 32 wherein cellulose sulfate is administered vaginally.
 34. Themethod according to claim 33 wherein said papilloma virus infectionresults in a malignant epithelial lesion.
 35. The method according toclaim 34 wherein said malignant epithelial lesion is cervical cancer.36. The method according to claim 23 wherein the sulfated polysaccharideis dextran sulfate.
 37. The method according to claim 36 wherein dextransulfate has an average Mr greater that about 500,000 daltons.
 38. Themethod according to claim 36 wherein the effective amount is about 0.1to 200 mg/ml.
 39. The method according to claim 38 wherein the effectiveamount is about 1 to 100mg/ml.
 40. The method according to claim 39wherein the effective amount is about 50 to 100 mg/ml.
 41. The methodaccording to claim 36 wherein dextran sulfate is administered incombination with a pharmaceutically acceptable carrier or diluent. 42.The method according to claim 41 wherein dextran sulfate is administeredvaginally.